The long-term goal of Project 4 is to develop an effective, clinically feasible approach to targeting chemoresistant tumor-initiating cells in chronic lymphocytic leukemia (CLL). In the preceding SPORE funding period, we showed that a small subset of circulating or marrow-resident CLL cells within the side population (SP) of tumor cells are drug resistant and have properties associated with cancer initiation. After demonstrating that a tumor vaccine consisting of autologous tumor cells modified to express IL-2 and CD40L could generate a T cell response (vaccine-induced cytotoxic T cells, or VICTs), directed primarily against the SP subset of CLL cells, we now propose to use this information to prepare T cells directed against the SP-associated antigens we previously identified, using ex vivo peptide immunization (Peptide-induced Cytotxic T cells- PICTs). We will also test a strategy for enhancing these T cell responses against the drug resistant and putative tumor initiating SP cells, by genetically modifying the SP-directed T cells to express IL-21, thus rendering them resistant to Treg cell inhibition. We will also administer the modified T cells together with continued booster vaccination with peptides to increase T cell expansion and persistence in vivo. These new strategies will be tested in NSG mice engrafted with CLL cells (Alms 1 and 2). In Aim 3 we plan to conduct a clinical trial (Aim 3) that will evaluate the most successful approach emerging from the CLL mouse model. The outcome of this project should provide a new and effective means of targeting, chemoresistant CLL-initiating cells, hence complementing extant methods of eradicating bulk populations of non-SP tumor cells. More broadly, it will confimi the existence of clinically relevant subset of CLL progenitors and demonstrate the feasibility of using immunotherapy to target these cells.